Porous ferro-or ferrimagnetic glass particles for isolating molecules

ABSTRACT

Porous, ferro- or ferrimagnetic, glass particles are described that selectively bind molecules of interest, especially nucleic acid molecules, under appropriate conditions. Methods of preparing the porous, ferro- or ferrimagnetic, glass particles and their use of identifying or separating molecules of interest are also described. Kits comprising the porous, ferro- or ferrimagnetic, glass particles are also provided.

[0001] In recent years, magnetic particles of various compositions andproperties have become available to facilitate purification, separation,and assay of various molecules. Magnetic particles or beads that bind amolecule of interest can be collected or retrieved by applying anexternal magnetic field to a vessel containing the particles. Unboundmolecules and supernatant liquid can be separated from the particles ordiscarded, and the molecules bound to the particles may be eluted in anenriched state. Thus, magnetic particles offer the potential for arelatively rapid, easy, and simple means to purify or separate moleculesof interest from a liquid phase or a mixture of other molecules.Furthermore, magnetic particles that bind specific molecules may beintegrated into robotic, multi-well, or multiplex sample assays orscreening systems to rapidly and automatically assay or identifymolecules of interest out of hundreds or even thousands of samples. Suchsystems are finding increasingly more applications in the purificationor isolation of biomolecules, such as nucleic acids and protein.Accordingly, a magnetic particle with an increased capacity to bind andisolate molecules, especially biomolecules, of interest would serve as avaluable tool in a variety of separation or isolation applications,including analytical and preparative procedures, as well as inmechanized systems designed to automatically screen arrays of hundredsor even thousands of samples for a particular molecule or class ofmolecules of interest.

[0002] The invention provides highly porous, ferromagnetic orferrimagnetic, glass (silica) particles that exhibit high bindingcapacities for molecules of interest, especially biomolecules, and, mostpreferably, nucleic acid molecules. The porous, ferro- or ferrimagnetic,glass particles of the invention can bind molecules, especially nucleicacid molecules, in a mixture, and then be collected or retrieved whilestill retaining the bound molecules by applying an external magneticfield to a side of a vessel containing the mixture and the magneticparticles or by inserting a magnetic probe into the vessel. The boundmolecules may then be eluted from the magnetic particles in a purerstate and in useful amounts owing to the high binding capacity of themagnetic particles of the invention for the molecules of interest.

[0003] The porous, ferro- or ferrimagnetic, glass particles of theinvention comprise silicon dioxide (SiO₂) and iron oxide particles orpigments. The iron oxide particles or pigments may be, e.g., Fe₂O₃(hematite), Fe₃O₄ (magnetite), or a combination thereof. Preferably, theiron oxide is ferrimagnetic magnetite.

[0004] In another embodiment of the invention, the porous, ferro- orferrimagnetic, glass particles of the invention have a composition thatis about 30-50% (by weight) Fe₃O₄ and about 50-70% (by weight) SiO₂.More preferably, the composition of the porous, magnetic, glassparticles described herein is about 35-45% (by weight) Fe₃O₄ and about55-65% (by weight) SiO₂.

[0005] In yet another embodiment, one or more oxides of other metals ortransition metals may also be present in the porous, magnetic, glassparticles of the invention. Such additional metal oxides may provideadditional desirable properties to the porous, magnetic, glassparticles. Preferably, an additional metal oxide is selected from thegroup consisting of oxides of titanium, boron, sodium, potassium,magnesium, calcium, zinc, lead, and combinations thereof.

[0006] The porous, magnetic, glass particles of this invention showferro- or ferrimagnetic behavior due to the presence of iron oxides oriron bearing pigments. If an external magnetic field is applied, theyare magnetized and remain magnetized (remanence) even when the externalmagentic field is removed, but this remaining magnetism is too weak toagglomerate or aggregate the particles.

[0007] In another aspect of the invention, the porous, magnetic, glassparticles described herein have an average size range of about 5-25 μm,preferably about 6-15 μm, and, most preferably, about 7-10 μm indiameter. Preferably, the total surface area of the porous, magnetic,glass particles of the invention, as measured by the nitrogen BrunaurEmmet Teller (BET) method, is 190 m²/g or greater and, more preferably,in the range of about 190-270 m²/g. Preferably, the porous, magnetic,glass particles of the invention have a cumulative pore area for poresgreater than 10 nm in diameter, as measured by the mercury(Hg)-porosimetry method, that is in the range of about 4-8 m²/g.

[0008] In another aspect of the invention, methods for manufacturingporous, magnetic, glass particles with high binding capacities fornucleic acids, or other biomolecules, are provided. A preferredmanufacturing process of the invention comprises providing a suspensionof magnetic iron oxide particles or pigments having an average size of75-300 nm in diameter. More preferably, 80% or more, and even morepreferably, 90% or more, of the iron oxide particles are 75-300 nm indiameter. Preferably, the iron oxide particles are suspended in glycerolor glycol, and combined with a source of silica (glass), and preferablyat a pH in the range of 6 to 8, and more preferably pH 7. Silica is thensynthesized in the presence of the iron oxide particles by hydrolyzingthe source of silica with acidic or alkaline buffer so that the silicaprecipitates or adsorbs on the surface of the iron oxide particles. Thesilica-coated iron oxide particles are allowed to aggregate to formlarger, porous, magnetic, glass particles. The nascent porous, magnetic,glass particles are then dried using an oven at a temperature below theCurie temperature. More preferably, the drying temperature is betweenabout 100° C. and about 500° C., such as 200° C. or 300° C. Even morepreferably, the drying temperature is between about 300° C. and about500° C.

[0009] In another embodiment of the manufacturing methods of theinvention, the source of silica is a tetraalkoxysilane, a silyl ester ofa multifunctional alcohol, a silicate, such as sodium silicate, silicananoparticles, or combinations thereof. More preferably, the source ofsilica is a tetraalkoxysilane, and most preferably, thetetraalkoxysilane is tetraethoxysilane.

[0010] In yet another embodiment of the manufacturing process of theinvention, the source of silica is hydrolyzed in the manufacturingprocess using a buffer that has an acidic pH of 5 or lower or a bufferthat has an alkaline pH of 9 or higher. Preferably, the hydrolyzingbuffer is an ammonia/ammonium salt buffer having a pH of between 9 and11.

[0011] In a preferred embodiment, porous, ferro- or ferrimagnetic, glassparticles of the invention bind greater than 1 μg of nucleic acidmolecules per mg of particle, even more preferably about 1.3 μg ofnucleic acid molecules per mg of particle, and most preferably greaterthan 1.3 μg of nucleic acid molecules per mg of particle.

[0012] In another embodiment, the yields of nucleic acid moleculesisolated using the porous, ferro- or ferrimagnetic, glass particles ofthe invention are 80% or greater.

[0013] Another aspect of the invention is a method for isolating amolecule of interest from a mixture, comprising:

[0014] providing a mixture containing the molecule of interest;

[0015] contacting the mixture with porous, ferro- or ferrimagnetic,glass particles of the invention;

[0016] allowing the molecule of interest in the mixture to bind oradhere to the porous, ferro- or ferrimagnetic, glass particles;

[0017] collecting the porous, ferro- or ferrimagnetic, glass particlescontaining the adherent molecule of interest by applying an externalmagnetic field; and

[0018] separating the porous, ferro- or ferrimagnetic, glass particleswith the adherent molecule of interest from the unbound components ofthe mixture.

[0019] Optionally, the bound molecule of interest may be eluted from theparticles of the invention by using an appropriate elution buffer.

[0020] In a preferred embodiment of the methods of isolating orseparating a molecule of interest from a mixture using the porous,ferro- or ferrimagnetic, glass particles of the invention, the moleculeof interest is selected from the group consisting of nucleic acids,proteins, polypeptides, peptides, carbohydrates, lipids, andcombinations thereof. More preferably, the molecule of interest is anucleic acid molecule, which may be any nucleic acid molecule, includingplasmid DNA, genomic DNA, cDNA, polymerase chain reaction-generated DNA(PCR DNA), linear DNA, RNA, ribozymes, aptamers, and chemicallysynthesized nucleic acid molecules.

[0021] In another embodiment, the invention provides kits for isolatingor separating molecules of interest, preferably nucleic acid moleculesof interest, comprising porous, ferro- or ferrimagnetic, glass particlesof the invention. A kit of the invention may further comprise one ormore buffers or concentrated stock solutions for suspending and usingthe porous, magnetic, glass particles of the invention. A buffer in akit of the invention may also contain one or more chaotropic agents,such as guanidinium isothiocyanate.

[0022] The porous, ferro- or ferrimagnetic, glass (silica) particlesdescribed herein have a relatively high binding capacity for variousmolecules, and especially nucleic acids, such that the particles areuseful in isolating or separating molecules from a mixture in usefulyields. The particles may be used in both analytical as well aspreparative scale procedures. Particles having a particular porosity,binding capacity, and binding specificity are obtained by selectivelychanging various synthetic reaction parameters according to theinvention.

[0023] In order that the invention may be more fully understood, thefollowing terms are defined.

[0024] “Pore”, as understood and used herein, refers to any inlet,depression, or recess in the outer surface of a particle in which thedepth of the depression or recess extends beyond the length of theradius of the inlet, depression, or recess measured at the surface ofthe particle. Inlets, depressions, or recesses that do not extend deeperthan the radius at the outer surface of the particle are not pores.

[0025] “Micropore”, as understood and used herein, refers to any porethat has an average diameter of less than 2 nm.

[0026] “Mesopore”, as understood and used herein, refers to any porethat has an average diameter in the range of 2 nm-200 nm.

[0027] “Macropore”, as understood and used herein, refers to any portthat has an average diameter of greater than 200 nm.

[0028] “Diameter of a pore”, as understood and used herein, refers tothe diameter of the pore at the narrowest point of the respective pore.

[0029] “Size of a particle”, as understood and used herein, refers tothe diameter of a particle. For a spherical particle, the sizecorresponds to its diameter. More generally, the size of regularly orirregularly shaped particles refers to the projected area of thediameter of the particle, expressed by the diameter of a circle with thesame area as that of the particle resting in a stable position.

[0030] “Cumulative pore area”, as understood and used herein, refers tothe calculated total pore area of pore walls, for pores having a certaindiameter (size).

[0031] “Surface area”, as understood and used herein, refers to thesurface area of a porous particle, which is equal to the sum of itsinner and outer surface areas.

[0032] “Outer surface” of a particle, as understood and used herein,refers to each and every point of a particle from which a line that isperpendicular to that point is able to extend outward withoutintersecting another portion of the particle.

[0033] “Inner surface”, of a porous particle, as understood and usedherein, refers to the surface that originates from the pore walls.

[0034] “Paramagnetic” substances, as understood and used herein, exhibita weak magnetic property only in the presence of an applied magneticfield. In the absence of an applied magnetic field, the spin and orbitalmoments are unaligned; pointing randomly to cancel each other out.However, in the presence of an externally applied magnetic field, spinand orbital moments tend to turn toward the direction of the field.However, thermal agitation of atoms of paramagnetic substances opposesthe tendency for all the magnetic moments to align. The result is onlypartial alignment of the moments in the direction of the appliedmagnetic field. As long as the magnetic field is applied, the substancewill exhibit a net, but relatively weak, magnetic field. When theexternal magnetic field is removed, the partial alignment deteriorates,and no magnetic field survives in the substance.

[0035] Like paramagnetic substances, “superparamagnetic” substances, asunderstood and used herein, also exhibit an induced and temporarymagnetic field in the presence of an externally applied magnetic field.In truly superparamagnetic substances, the magnetic moments ofindividual atoms of the substance are able to align and add up to form amuch stronger induced magnetism than is possible in a paramagneticsubstance (see, for example, Bean and Livingstone, J. Appl. Physics, 30:120S-129S (1959)). Thus, the induced magnetic field of superparamagneticsubstances is significantly stronger (by several orders of magnitude)than the fields generated in substances classically defined as“paramagnetic”. Iron oxide crystals of less than about 300 angstroms (30nm) in diameter are capable of exhibiting such superparamagneticbehavior.

[0036] “Ferromagnetic” substances, e.g., hematite (Fe₂O₃), F_(Metal),Ni, Co, as understood and used herein, are substances that are capableof exhibiting a magnetic field even in the absence of an appliedmagnetic field. In ferromagnetic substances, regions or domains of thesubstance are capable of aligning magnetic moments in the same directionresulting in a magnetic field. If an external magnetic field is appliedto a ferromagnetic substance, the various domains of the substance canbecome aligned in the same direction to yield a very strong magneticfield in the ferromagnetic substance. Even if the external magneticfield is removed, the domains of the ferromagnetic substance tend toremain aligned in the same direction and so the substance as a wholeretains a strong magnetic field, essentially unperturbed by any innatethermal agitation. However, by heating a ferromagnetic substance to asufficiently high temperature, thermal energy can exceed themagnetization energy so that the alignment of magnetic momentsdeteriorates and becomes random. At such temperatures, the substance iscapable of exhibiting a paramagnetic behavior in the presence of anexogenously applied magnetic field. The temperature at which aferromagnetic substance becomes paramagnetic is known as the “Curietemperature” or “Curie point”.

[0037] “Ferrimagnetic” substances, as understood and used herein,exhibit a magnetic field that is retained (remanence) after beingexposed to an externally applied magnetic field, similar toferromagnetic substances. Ferrimagnetism is the magnetic property thatis only found in ferrites, which are mixed oxides (M²⁺O)(Fe₂O₃), whereone cation is a divalent ion (M²⁺) from the group of transition metals,e.g., Fe²⁺, Mn²⁺, Zn²⁺, Co²⁺, etc., and the trivalent cation is Fe³⁺,e.g., magnetite (Fe₃O₄). The crystal structure of ferrites consists oftwo interlocking crystals. When the spins of the atoms of one latticeposition are aligned in a particular orientation and the spins of atomsin another position are aligned in an opposite orientation, a magneticfield will be retained (remanence). Thus, ferrimagnetic substances arecrystalline ferric oxide compounds, which resemble ferromagneticsubstances in their ability to retain a magnetic field in the absence ofan externally applied magnetic field.

[0038] The porous, magnetic, silica particles of this invention showferro- or ferrimagnetic behavior and remain magnetized even in theabsence of an external magnetic field, but this remaining magnetism istoo weak to agglomerate or aggregate the particles.

[0039] Unless noted otherwise, the terms “silica”, “silica glass”, and“glass”, as understood and used herein, refer to an amorphous,crystalline form of SiO₂ that covers all or a portion of the iron oxideparticles or pigments that are used to make the porous, ferro- orferrimagnetic, glass particles of this invention.

[0040] The porous, ferro- or ferrimagnetic, glass particles of theinvention are useful for isolating or separating nucleic acid moleculesfrom any mixture or sample. A “mixture” or “sample”, as understood andused herein, includes any mixture or preparation that contains amolecule of interest, whether the mixture is man-made or derived from anatural or biological source, such as cells, tissues, or viruses. Amixture or sample may be complex, e.g., containing many components inaddition to a molecule of interest, or relatively simple, such as anaqueous solution of a molecule of interest. A mixture includes, but isnot limited to, any of the various in vitro reaction mixtures used tomanipulate or synthesize nucleic acids or that contain nucleic acids,such as polymerase chain reaction (PCR), nucleic acid sequencingreactions, restriction endonuclease or other nuclease digestionreactions, nucleic acid hybridization assay mixtures, protein-nucleicacid binding assay mixtures, antibody-nucleic acid assay mixtures, andin vitro transcription and/or translation assay mixtures. A biologicalmaterial that may be in a mixture or sample includes, but is not limitedto, blood, plasma, lymph, milk, urine, semen, or other biologicalfluids, whole cells, extracts of cells, viral particles, hair, andtissue homogenates.

[0041] Compositions and Methods of Manufacturing Porous, Magnetic, GlassParticles

[0042] The porous, ferro- or ferrimagnetic, glass particles of thisinvention contain iron oxide and silica glass. Preferably, the particleshave a relatively simple composition that is 30-50% (by weight) Fe₃O₄and 50-70% (by weight) SiO₂. More preferably, the particles of thisinvention are 35-45% (by weight) Fe₃O₄ and 55-65% (by weight) SiO₂.However, other compounds may be incorporated into the reaction mixturesto obtain particles having properties that are better suited for aparticular protocol. Accordingly, in addition to iron oxide, theparticles of this invention may also contain oxides of other metals,especially transition metals, and include, without limitation, oxides oftitanium, boron, sodium, potassium, magnesium, calcium, zinc, and lead.Preferably, iron oxide is the most prevalent metal oxide by weight inthe particles of this invention.

[0043] The size of the iron oxide particles or pigments used in themanufacturing process affects the size and characteristics of the finalparticle product. For this reason, the component iron oxide particles orpigments preferably have an average size in the range of about 75 to 300nm in diameter. More preferably, at least 80% of the iron oxideparticles have an average size in the range of about 75 to 300 nm indiameter. Even more preferably, at least 90% of the iron oxide particleshave an average size in the range of about 75 nm to 300 mn in diameter,and, most preferably, at least 95% of the iron oxide particles used tomake the porous, magnetic glass particles of this invention are in thissize range.

[0044] The pores of the porous, magnetic, glass particles describedherein are present in a wide range of sizes as determined by thediameter of the pores at the outer surface of the particles. The highpore content of the particles of the invention, is also appreciated bythe fact that particles of the invention, which are preferably in therange of about 5 to 25 μm in diameter, more preferably about 6 to 15 μmin diameter, and most preferably, about 7 to 10 μm in diameter, alsohave relatively high values for the BET specific surface area of 190m²/g or greater and, preferably, in a range of 190-270 m²/g; indicativeof a high pore surface area for particles of this size. Thus, poresclassified as micropores, mesopores, and macropores may be allrepresented on each particle prepared according to the invention.Notably, the pores present in the particles described herein includepores with diameters greater than and less than 10 nm in diameter. Thehigh porosity of the porous, magnetic, glass particles of the inventionis also appreciated by the fact that the cumulative pore area of theparticles as determined by standard mercury porosimetry for poresgreater than 10 nm in diameter is typically greater than 4 m²/g and,preferably, in the range of about 4 to 8 m²/g.

[0045] The iron oxide particles or pigments constitute the basicmagnetic nuclei of the porous, magnetic, glass particles of theinvention. As silica is deposited or precipitated on to the iron oxideparticles during the manufacturing process, the iron oxide particlesbegin to aggregate to form the larger, porous, magnetic, glass particlesof the invention which, as noted above, have large surface areas. Unlessalready available in the preferred average size range of about 75 to 300nm in diameter, the iron oxide particles or pigments may have to bereduced to the preferred size prior to carrying out the manufacturingprocess. The iron oxide particles or pigments may be processed to thepreferred average range of sizes of about 75 nm to about 300 nm indiameter using any of a variety of methods known in the art. Forexample, the iron oxide particles or pigments may be ground down to anappropriate size using a ball mill, such as a PM 400 planetary ball mill(Retsch, Haan, Germany). More preferably, the iron oxide particles areground by rapid stirring, for example, by using a commercially availablestirrer for laboratory use. The iron oxide particles or pigments shouldbe suspended in an aliphatic C₁-C₆-alcohol, more preferably, analiphatic C₁C₄-alcohol, such as isopropanol, ethanol, glycol, orglycerol. Preferably, glycerol is used because its higher viscosityallows preparation of smaller iron oxide particles. Most preferably, theiron oxide particles are ground to the average size range of about 75 nmto about 300 nm by rapid stirring in a solution of glycerol (e.g., 43%glycerol).

[0046] The silica component of the porous, magnetic, glass particlesdescribed herein is generated during the manufacturing procedure from atetraalkoxysilane, preferably having the formula Si(OC_(n)H_(2n+1))₄,where n is an integer of 1-5. This silica synthesis step may also usesilyl esters of multifunctional alcohols, such as glycerol and glycol.In another embodiment of the invention, the silica source may be asilicate, more preferably, a sodium silicate or silica nanoparticles.Alternatively, the source of silica may be a combination of at least onetetraalkoxysilane and at least one silyl ester of a multifunctionalalcohol. Most preferably, the source of silica for the porous, magnetic,glass particles of this invention is tetraethoxysilane.

[0047] In a preferred embodiment of the manufacturing procedure,tetraalkoxysilane is added to a suspension of ferro- or ferrimagneticiron oxide particles, as a solution of tetraethoxysilane (30%) in analiphatic C₁-C₆ alcohol, more preferably an aliphatic C₁-C₄ alcohol,(70%) at a pH between 6 and 8, more preferably pH 7.

[0048] During the process of manufacturing the porous, magnetic, glassparticles of the invention, tetraalkoxysilane, silyl ester of amultifunctional alcohol, or a combination thereof, is hydrolyzed torelease silica by changing the pH of the reaction mixture to an acidicpH, e.g., pH 6 and lower, or to an alkaline pH, e.g., pH 9 and higher.Acidic buffers that may be used to hydrolyze a tetraalkoxysilaneinclude, but are not limited to, acetate buffers that have a pH of 5.Alkaline buffers that may be used to hydrolyze the tetraalkoxysilane inthe methods of the invention, include but are not limited toammonia/ammonium salt buffers (for example, ammonia/ammonium chloridebuffer) that have a pH in the range of 9 to 11. Preferably, silica issynthesized using an alkaline buffer to hydrolyze the tetraalkoxysilaneto release silica. More preferably, the silica is synthesized using anammonia/ammonium salt buffer at a pH of between 9-11.

[0049] In an other embodiment of the process of manufacturing theporous, magnetic, glass particles of the invention, a silicate or silicananoparticles are hydrolyzed to release silica by changing the pH of thereaction mixture to an acidic pH, e.g., pH 6 and lower. More preferably,the silica is synthesized using acetic acid.

[0050] The silica released upon hydrolysis of the tetraalkoxysilane orother source of silica precipitates, deposits, or adsorbs on the surfaceof the iron oxide particles or pigments, which then aggregate to formthe larger, porous, magnetic, glass particles of the invention.

[0051] To carry out the silica synthesis step, the iron oxide particlesor pigments may be mixed first with the source of silica and then thehydrolysis buffer of acidic or alkaline pH added, or the iron oxideparticles may be mixed first with the hydrolysis buffer and the silicasource added thereafter. However, the preferred procedure is to firstmix the iron oxide particles with the silica source, and, thereafter,add the hydrolyzing buffer. In addition, the silica synthesis step ispreferably carried out in a final reaction solution that is viscousenough to promote the production of small silica particles, which areable to more efficiently deposit or precipitate over the surface of theiron oxide particles. In a preferred embodiment, ammonia/ammoniumchloride buffer (5 M, pH 10.5) is added dropwise to iron oxideparticles, that are dispersed in a solution of glycerol (43%), ethanol(43%), and tetraethoxysilane, over a time period of ten (10) minuteswhile stirring the mixture at 2000 rpm. Adding the buffer over a periodof time shorter than 10 minutes tends to produce unmagnetic particles,whereas adding the buffer over a longer time period tends to produceparticles with a reduced porosity that are too compact and less thanoptimal for isolating or separating nucleic acid molecules from asample. Stirring at lower speeds, such as 500 rpm, during silicasynthesis results in porous, magnetic, glass particles that have lowerbinding capacities for nucleic acids.

[0052] As silica is synthesized by hydrolysis of tetraalkoxysilane orother silyl ester compound, it precipitates or adsorbs on the surface ofthe iron oxide particles. The silica containing iron oxide particleswill then aggregate to form larger, porous, magnetic, glass (silica)particles. It is recommended that the newly formed particles be allowedto further incubate (“age”) so that they may solidify or stabilize. Aneffective aging step may involve simply allowing the newly formedparticles to continue to incubate in the silica synthesis mixture withstirring for an additional 8 to 24 hours.

[0053] The stabilized, newly formed particles are separated from thereaction mixture by filtration and then washed with a solvent solution,usually an alcohol solution. The wash solution may contain othersolvents and agents in addition to or in place of alcohol, includingacetone and/or a chaotropic agent(s). In general, however, an anhydrousalcohol, especially absolute ethanol, alone is preferred for washing thenewly formed particles. An anhydrous alcohol, such as absolute ethanol,is highly effective at preventing agglomeration of newly formedparticles and in producing particles that have or retain a high bindingcapacity for nucleic acid molecules.

[0054] After filtration and washing, the newly formed porous, magnetic,glass particles can be dried. Optimal drying temperatures are alwaysbelow the Curie temperature and may be as low as about 100° C. or ashigh as about 500° C. Preferably, the temperature is in the range ofabout 300° C. to 500° C., such as 200° C. More preferably, optimalresults are obtained by drying the newly formed particles at about 300°C. in a circulating air, drying oven. The preferred average size of thefinal porous, magnetic, glass particles is about 5 to 25 μm, morepreferably about 6 to 15 μm, and most preferably, about 7 to 10 μm indiameter. The dried particles may be stored in an enclosed vessel atroom temperature for months without showing signs of aging, i.e.,deterioration of any properties of the particles.

[0055] Another method of making the ferro- or ferrimagnetic particlesaccording to the invention comprises the steps of providing a suspensionof ferro- or ferrimagnetic iron oxide particles in alcohol, preferablyethanol, adding silica nanoparticles to the suspension of the ferro- orferrimagnetic iron oxide particles at a pH lower than 6, aging themixture by continuous stirring, separating, e.g., magnetically, theresulting porous, ferro- or ferrimagnetic, glass particles from theliquid, washing the separated porous, ferro- or ferrimagnetic, glassparticles, and drying the porous, ferro- or ferrimagnetic, glassparticles at a temperature at about 200° C.

[0056] The following guidelines are recommended to produce highlyporous, magnetic, glass particles that exhibit high binding capacitiesfor nucleic acid molecules. Preferably, the iron oxide particles orpigments used in the synthesis of the porous, magnetic, glass particlesof the invention are ferrimagnetic magnetite (Fe₃O₄) particles. The ironoxide particles or pigments should have an average size in the range of75 nm to 300 nm in diameter. It may be necessary to reduce the size ofthe iron oxide particles or pigments to this recommended size range bygrinding. Although ball milling may be used to grind the iron oxideparticles or pigments down to the recommended size range, the grindingstep preferably is carried out by rapid stirring, such as at 2000 rpm,and in a viscous solution, such as a glycerol solution. Silica issynthesized by hydrolysis of a tetraalkoxysilane, silyl ester, orsilicate, or by precipitation of silica nanoparticles using an acidic oralkaline buffer. Tetraethoxysilane is the preferred source of silica forsynthesizing the particles of the invention. The silica synthesis shouldbe carried out in a viscous solution, for example, a solution containingglycerol, to promote synthesis of small silica particles, which are ableto more efficiently cover or precipitate on the surface of the ironoxide particles or pigments. Preferably, the hydrolysis buffer is addedto the iron oxide particles suspended in a viscous tetraalkoxysilane orsilyl ester compound mixture. Furthermore, the source of silica,especially if the source is tetraethoxysilane, should be added over aperiod of ten (10) minutes. Anhydrous alcohol, especially absoluteethanol, is the preferred solvent for washing the newly formed porous,magnetic, glass particles. The newly formed, porous, magnetic, glassparticles should be aged to allow them to stabilize. Finally, the newlyformed particles should be dried at relatively high temperatures, butbelow the Curie temperature, in an oven between 100° C. and 500° C.,preferably between 300° C. and 500° C., and even more preferably at 300°C.

[0057] Porous, ferro- or ferrimagnetic, glass particles of the inventionmay also be provided in a kit for separating, detecting, or isolatingany of variety of molecules of interest in a mixture including, but notlimited to, nucleic acids, proteins, polypeptides, peptides,carbohydrates, lipids, and combinations thereof. Preferably, the kits ofthe invention comprise porous, ferro- or ferrimagnetic, glass particlesdescribed herein for separating, detecting, or isolating nucleic acidmolecules of interest or molecules containing nucleotides in a mixture.The kits of the invention may also include one or more buffers that areuseful for suspending the porous, magnetic, glass particles and/or forlater steps in the isolation or purification procedure for the nucleicacid or other molecule of interest. One or more buffers that may beincluded in the kits of the invention may contain one or more chaotropicagents or substances. According to this invention, preferred chaotropicagents include, without limitation, salts from the group oftrichloroacetates, thiocyanates (including guanidinium (or “guanidine”)isothiocyanate), perchlorates (such as sodium perchlorate), iodides(such as sodium iodide, potassium iodide), guanidinium hydrochloride,and urea. The chaotropic agents are preferably used in a range of 1 to 8M aqueous solution, more preferably in a range of 2 to 5 M aqueoussolution, and most preferably in a range of 2 to 4 M aqueous solution.Most preferably, the chaotropic agent in a buffer of a kit of theinvention is guanidinium isothiocyanate.

[0058] Use in Isolating Nucleic Acids and Other Biomolecules

[0059] The porous, magnetic, glass particles described herein have ahigh capacity to bind nucleic acids, thereby malting the particles ofthis invention especially useful for isolating or separating nucleicacid molecules from various samples and mixtures. The particles may alsobe used to isolate or separate other molecules including, but notlimited to, carbohydrates, polypeptides, peptides, lipids, and evencombinations of such molecules, such as glycoproteins and nucleicacid/protein combinations or assemblies. The selective isolation orseparation of one molecule over another may be achieved by adjusting thebuffer conditions at which a molecule of interest binds or elutes fromthe porous, magnetic, glass particles described herein. The magneticproperty of the porous, magnetic, glass particles of the inventionpermits the particles to be easily and rapidly collected from a sampleor mixture by applying an external magnetic field using any of a varietyof magnetic collection systems available in the art.

[0060] For example, the porous, magnetic, glass particles of theinvention may be used to isolate or separate any of a variety of nucleicacid molecules including, but not limited to, cDNA, PCR reactionproducts, plasmids, genomic nucleic acids, nucleic acid primers, variousspecies of RNA, ribozymes, aptamers, nucleic acid molecules containingsynthetically produced nucleotides, chemically synthesized nucleicacids, nucleic acid-protein complexes, hybridized nucleic acidmolecules, nucleic acid molecules in in vitro transcription and/ortranslation assays, and immunoassays, such as ELISA or radioimmuneassays, where such procedures contain a nucleic acid component.Synthetically produced nucleotides include nucleotides that haveconstituent moieties, i.e., sugar, nitrogenous heterocyclic base (purineor pyrimidine), and phosphate backbone, that are found in nature as wellas nucleotide compounds that have a constituent moiety that has beenmodified or substituted with a group not found in nature. For example,the particles of the invention may be used to isolate a nucleic acidmolecule that contains a synthetic nucleotide in which a thiol groupreplaces one or more phosphate groups, a modified purine or pyrimidinereplaces a naturally occurring purine or pyrimidine, or a differentmolecule replaces the ribose or 2-deoxyribose sugar moiety. Theparticles described herein also may be used to effectively stop areaction that depends on the presence of a nucleic acid molecule bybinding and separating the nucleic acid from the reaction. In addition,the particles described herein may be used to collect or scavengevaluable or hazardous nucleic acid molecules, for example, as may be thecase in forensic samples, archeological samples, accidental spills, andbreaches in containment vessels.

[0061] The porous, magnetic, glass particles of the invention may beused to preferentially separate a particular class or size of nucleicacid molecules from other nucleic acid molecules by adjusting steps inthe isolation procedure (see, below, Examples 6 and 7). Basic protocolsfor binding nucleic acids to magnetic particles have been described(see, e.g., PCT publication No. WO 95/01359, incorporated herein byreference). For example, nucleic acids may be isolated with the porous,magnetic, glass particles of the invention in the presence of salts inhigh concentrations that promote binding of the nucleic acid to theparticles. Preferably, one or more chaotropic agents (as describedabove) is also present, such as sodium perchlorate, guanidiniumhydrochloride, guanidinium isothiocyanate, potassium iodide, potassiumthiocyanate, sodium chloride, sodium isothiocyanate magnesium chlorideor sodium iodide. More preferably, the chaotropic agent is guanidiniumisothiocyanate. A chaotropic agent is used at a concentration that is,preferably, in the range of 1 to 8 M; more preferably, in the range of 2to 5 M; and, most preferably, in the range of 2 to 4 M. Furthermore, aC₁-C₅ aliphatic alcohol, such as methanol, ethanol, n-propanol,isopropanol, n-butanol, tert-butanol, n-pentanol, or combinationsthereof, in a concentration of 1 to 80% (vol/vol) may also be added tothe solution of chaotropic agent. Especially preferred is the use ofisopropanol.

[0062] The concentrations of salts and/or alcohols may be adjusted sothat nucleic acid molecules are bound selectively to the magneticparticles. Furthermore, it is possible to separate nucleic acids ofdifferent lengths from each other by adjusting the concentrations ofchaotropic salts and/or alcohols. Examples of various buffer conditionsfor binding and eluting nucleic acid molecules of interest to porous,magnetic, glass particles of the invention are described below (see,Examples).

[0063] The magnetic particles to which particular nucleic acid moleculesof interest are bound or adsorbed can be retrieved or separated from amixture magnetically. For example, the particles can be attracted to thewall of the vessel containing them by applying an external magneticfield, and the contents not bound to the particles can then be removed,e.g., by pipetting, decanting, or suction filtration. In an alternativeprocedure, the magnetic particles with the bound nucleic acid moleculesmay be separated from the unbound components of a mixture by immersing amagnet into the mixture to collect the particles containing the boundnucleic acid molecules, transferring the particles on the magnet toanother vessel, and, if desired, wiping or washing off the particlesfrom the magnet into the vessel and removing the magnet stripped of thecollected particles.

[0064] After the binding step, the particles may also be separated fromimpurities, if necessary, by washing steps with solvents and subsequentmagnetic separations. The wash solution may contain alcohols, otherhighly volatile organic solvents such as acetone, and even one or morechaotropic agents.

[0065] If it is appropriate for further utilization of the separatednucleic acid molecules, the nucleic acid molecules may be eluted with anelution buffer from the magnetic particles. The elution buffer maycontain, e.g., deionized water, aqueous solutions of low saltconcentrations, Tris-[hydroxymethyl]amino methane (Tris) buffer, and/orethylenediaminetetraacetate (EDTA).

[0066] A more complete appreciation of the invention, alternate andobvious embodiments, and the advantages thereof can also be obtainedfrom the following non-limiting examples.

EXAMPLES Example 1

[0067] Synthesis I: Synthesis of Porous, Magnetic, Glass Particles UsingAlkaline Hydrolysis of Tetraethoxysilane

[0068] Iron (II, III) oxide particles (Aldrich, Steinheim, Germany,catalog No. 31,006-9) were placed in a ball mill, such as a PM 400planetary ball mill (Retsch, Haan, Germany), and suspended in 50 ml ofisopropanol or ethanol. The mill jar was loaded with 3 mm diameteryttrium oxide balls, and the mill was run for 3 hours at maximumintensity. The milled particles were removed, the yttrium oxideparticles separated according to manufacturer's procedure, and 4 g ofmilled iron oxide particles were transferred to a 500 ml Erlenmeyerflask fitted with a reflux condenser and a stirrer (Merck GmbH; Koeln,Germany; catalog No. 9.197215). 150 ml of absolute ethanol and 45 ml of5 M ammonia/ammonium chloride buffer (pH 11) were added to the milledparticles in the flask and mixed by stirring at 500 rpm. 100 ml of atetraethoxysilane solution (30 ml tetraethoxysilane: 70 ml ethanol) wereadded dropwise with a peristaltic pump over a period of three hours atroom temperature, and the reaction mixture allowed to incubate (age) foranother 24 hours with continual stirring. The mixture was thensuction-filtered through a porosity 3 glass frit to collect theresulting particles. The collected particles were washed twice with 100ml deionized water, twice with 100 ml absolute ethanol, and twice morewith deionized water. The particles were then dried at 120° C. in acirculating air drying oven.

Example 2

[0069] Synthesis II: Synthesis of Porous, Magnetic, Glass ParticlesUsing Acid Hydrolysis of Tetraethoxysilane

[0070] Ten grams of Magnetic Pigment 345 (BASF, Ludwigshafen, Germany)were placed in a plastic vessel and mixed with 100 ml absolute ethanol.A homogenizer stirrer (Welabo, Duesseldorf, Germany; catalog No.333611312) was introduced into the plastic vessel, and the mixture wasstirred at about 1000 rpm for 3 hours, while the plastic vessel was keptcool with an ice bath. The mixed suspension and a 100 ml ethanol rinseof the plastic vessel were transferred to a 2 liter three-neck flask,and an additional 200 ml of ethanol are added. 100 ml of a 2 M aceticacid/acetate buffer (pH 4.0) were then added to the flask. The mixturewas stirred at 1000 rpm while 100 ml of a tetraethoxysilane solution (50ml tetraethoxysilane:50 ml ethanol) were added dropwise with a droppingfunnel over a period of 10 minutes. The mixture was allowed to ageovernight (approximately 8-12 hours) with continual stirring, and thenfiltered by suction through a porosity 3 glass frit to collect theresulting particles. The resulting particles were washed twice with 100ml of deionized water, twice with 100 ml absolute ethanol, and twiceagain with 100 ml of deionized water. The particles were dried for 8hours at 120° C. in a circulating air dry oven. The resulting particleshad a mean diameter of 10 μm.

Example 3

[0071] Synthesis III: Synthesis of Porous, Magnetic, Glass Particlesfrom Magnetite and Sodium Silicate

[0072] Ten grams of Magnetic Pigment 345 (BASP, Ludwigshafen, Germany)were suspended in 100 ml paraffin oil in a plastic vessel and stirredwith a homogenizing stirrer at 1000 rpm for 3 hours while the plasticvessel is kept cool with an ice bath. Then, 100 ml of paraffin oil wereadded to the plastic vessel, and the mixture was stirred again untilhomogenous. The mixture was transferred to a 2 liter three-necked. Anadditional 200 ml of paraffin oil were added, then 200 ml of 1-hexanol(Fluka, catalog No. 52840), and 60 ml of an aquaeous sodium silicatesolution (27% SiO₂ in water, Fluka, catalog No. 71957). The mixture wasthen stirred at 2000 rpm, while 60 ml of concentrated acetic acid wasadded dropwise over a period of 5 minutes. The mixture was then stirredfor an additional 60 minutes. Then the mixture containing the newlyformed particles was poured into centrifugation tubes, and the tubescentrifuged for 1 hour at 4000 rpm to collect the particles. Thesupernatant liquid is decanted, and the particles are suspended in amethanol solution (50%). The suspension of particles was suctionfiltered through a porosity 3 glass frit. The collected particles arethen washed twice with 100 ml of absolute ethanol, and then twice with100 ml of deionized water. The particles were then dried in acirculating air drying oven at 200° C. The resulting porous, magnetic,glass particles had a mean diameter of 25 μm.

Example 4

[0073] Synthesis IV: Synthesis of Porous, Ferrimagnetic, SilicaParticles

[0074] In a 500 ml plastic vessel, 200 ml anhydrous glycerol was added,and the vessel adjusted under a stirrer. 24 g magnetite (Bayoxide 8713H, manufactured by Bayer AG, Leverkusen, Germany) was added slowly tothe glycerol with slow stirring, and stirring was continued for twohours at 2,000 rpm to destroy agglomerations. Then, the stirring speedwas reduced and 250 ml glycerol was added to the suspension. After fiveminutes, the reaction mixture was transferred to a 4 liter flask withstirrer (see, Example 1) and dropping funnel. 450 ml glycerol, 900 mlethanol, and 120 ml tetraethoxysilane were added, and the stirring speedadjusted to 2,000 rpm. Within ten minutes, 300 ml of a 7 M ammoniumchloride buffer, pH 10.5, were added, and the stirring speed wasmaintained for twelve hours. Then, the reaction mixture was filtered,washed two times with demineralized water and two times with ethanol anddried for seven hours at 300° C. The particles had a particle size of 5to 10 μm.

Example 5

[0075] Synthesis V: Synthesis of Porous, Magnetic, Glass Particles fromMagnetite and Silica Nanoparticles

[0076] Ten grams of Magnetic Pigment 345 (BASF, Ludwigshafen, Germany)and 100 ml of absolute ethanol were placed in a plastic vessel andstirred with a homogenizing stirrer for 2 hours at 1000 rpm. Anadditional 100 ml of ethanol were added, and the mixture stirred for 5minutes more. The contents of the vessel were transferred to a 1 literthree-necked flask. 30 ml of LUDOX AS40 (Aldrich, Deisenhofen, Germany,catalog No. 42,084-0) and 400 ml of deionized water were also added tothe flask. The mixture in the flask was stirred at 1000 rpm for 5minutes. Then 50 ml of concentrated acetic acid were added dropwise overa period of 5 minutes with continual stirring. The mixture was stirredfor an additional 30 minutes at 1000 rpm and then at 500 rpm for afurther 60 minutes. The mixture containing newly formed particles wassuction filtered through a porosity 3 glass frit to collect theparticles. The collected particles were washed twice with 100 ml ofdeionized water, twice with 100 ml of absolute ethanol, and twice morewith 100 ml of deionized water. The suction was continued until thewashed particles were dry. The particles were further dried for 7 hoursat 200° C. in a circulating air drying oven. The porous, magnetic, glassparticles produced by this procedure had an average diameter of 25 μm.

Example 6.

[0077] Use of Porous, Magnetic, Glass Particles to Purify Plasmid DNAfrom Smaller Nucleic Acid Molecules

[0078] This example compares the ability of three different magneticparticles to purify a plasmid DNA molecule from a mixture of shorter DNAmolecules. In this example, magnetic particles from three differentsources are used to separate a 3 kb plasmid vector from a mixture ofpolymerase chain reaction (PCR) oligonucleotide primers. The magneticparticles were obtained by the procedure in Example 4 as an example ofthe porous, magnetic, glass particles of the invention, from RocheDiagnostics (mRNA Isolation Kit, catalog No. 1934333, Roche Diagnostics,Mannheim, Germany), and from Promega (WIZARD PURE FECTION® Plasmid DNAPurification Systems, catalog No. A2150, Promega Corp., Madison, Wis.).

[0079] A solution of nucleic acid molecules was prepared containing thephagemid pBLUESCRIPT II, which is 2.96 kb (Stratagene GmbH, Heidelberg,Germany) and a set of commercially available oligomeric PCR primers (TIBMolbiol, Berlin, Germany), which have nucleotide lengths of 20nucleotides (20 mer), 45 nucleotides (45 mer), 56 nucleotides (56 mer),and 75 nucleotides (75 mer), at a concentration of 1 μg of oligomer per50 μl.

[0080] Three preparations of magnetic particles obtained from Example 4,were freed of buffer contaminants by magnetic separation, washed twotimes with water and two times with absolute ethanol, and vacuum dried.The particles were then suspended in buffer PB, a solution containingchaotropic agents, (QIAGEN Inc., Valencia, Calif., USA, catalog No.19066) at a concentration of 23.5 mg/ml. 100 μl of each suspension ofparticles were mixed with a 50 μl aliquot of the nucleic acid moleculessolution in a 500 μl PCR Eppendorf tube. The particles and nucleic acidmolecules were then mixed for 1 minute on an IKA Minishaker (IKA,Staufen, Germany). The PCR tubes were then placed in a Dynal MPC-P-12magnetic separator to collect and separate the magnetic particles fromthe liquid, and the supernatant liquid was then discarded. The particleswere then washed by resuspending four times in buffer PE (QIAGEN Inc.,Valencia, Calif., USA, catalog No. 19065).

[0081] The washed particles were dried for 15 minutes in a heating blockat 37° C. to remove residual ethanol. To elute nucleic acid moleculesfrom the particles, 30 μl of elution buffer (10 mM Tris HCl(Tris[hydroxymethyl]amino methane), pH 8.5) were added to the particlesin the PCR tubes, and the tubes were then mixed for 1 minute on an IKAMinishaker. The PCR tubes were then placed in the magnetic separator,and the particles separated magnetically. 25 μl of eluate were thenpipetted from the tubes. To analyze the yield of nucleic acid molecules,15 μl samples of the eluates were run on a standard 2% TAE (Tris,acetate, EDTA (ethylene diamine tetraacetate) agarose gel containingethidium bromide. Table 1 below shows the yields of the various nucleicacid molecules purified by the three types of magnetic particles. TABLE1 Yield of Yield of Yield of Yield of Yield of Source of plasmid 20mer45mer 56mer 75mer particle (percent) (percent) (percent) (percent)(percent) Example 4 88 0 4 5 6 Boehringer 53 11 37 47 32 Promega 80 0 2333 38

[0082] The yields of nucleic acid molecules in Table 1 clearly show thatthe porous, magnetic, glass particles produced by the procedure inExample 4 were more effective in purifying plasmid DNA from the smallernucleic acid molecules than either of the commercially availablepreparations of magnetic particles.

Example 7

[0083] Use of Porous, Magnetic, Glass Particles to Purify Genomic DNAfrom Whole Blood

[0084] This example demonstrates the use of porous, magnetic, glassparticles of this invention to purify genomic DNA from human bloodcells.

[0085] Porous, magnetic, glass particles were synthesized according tothe procedure in Example 4. A protease solution was prepared bydissolving 110 mg of QIAGEN protease (QIAGEN Inc., Valencia, Calif.,USA, catalog No. 19157) in nuclease free water containing 0.04% sodiumazide. This protease solution is stable for at least 2 months whenstored at 2-8° C. AW1 buffer was prepared by mixing 125 ml of absoluteethanol (Fluka AG, Buchs, Switzerland) with 95 ml of AW1 concentratedbuffer stock (QIAGEN Inc., Valencia, Calif., USA, catalog No. 19081).AW2 buffer was prepared by mixing 160 ml of absolute ethanol with 66 mlof AW2 concentrated buffer stock (QIAGEN Inc., Valencia, Calif., USA,catalog No. 19072). Porous, magnetic, glass particles (180 mg) weresuspended in 1 ml of a chaotropic AL buffer (QIAGEN Inc., Valencia,Calif., USA, catalog No. 19075), and the suspension mixed with ahomogenizer (IKA Minishaker) immediately before use.

[0086] The porous, magnetic, glass particles of the invention wereemployed to isolate genomic DNA from blood cells using the followingblood spin protocol.

[0087] 20 μl of the protease solution was transferred into the bottom ofa microcentrifuge tube and mixed with 200 μl of Buffer AL and with 200μl of whole human blood in a microcentrifuge by pulse-vortexing for 15seconds to obtain a homogenous mixture (cell extract). 20 μl of ahomogenous suspension of magnetic particles in AL buffer were added,followed by pulse-vortexing the microfuge tube for 15 seconds. Themixture of magnetic particles and cell extract was then incubated at 56°C. for 10 minutes. 250 μl isopropanol were added to the mixture, and themicrofuge tube was immediately pulse-vortexed for 15 seconds.

[0088] The microfuge tube containing the magnetic particles and cellextract was placed on a magnetic separator (Dynal AS, see above) toseparate the magnetic particles from the supernatant liquid, which wasthen discarded. The particles were washed twice with 500 μl of BufferAW1, by resuspending the particles in the buffer and vortexing brieflyto thoroughly suspend the particles. After each vortexing, the particleswere collected using the magnetic separator. In the same manner, theparticles were then washed twice with 500 μl of Buffer AW2. After thisset of washes, the final wash buffer was removed, and the tube wasallowed to set in the magnetic separator for 15 minutes at roomtemperature to permit the particles to dry and to remove residualethanol by volatilization.

[0089] After this drying period, the DNA bound to the magnetic particleswas eluted by suspending the particles in 100 μl of Buffer AE (QIAGENInc., Valencia, Calif., USA, catalog No. 19077), vortexing briefly tosuspend the particles, and incubating the suspension for 1 minute atroom temperature. The magnetic particles were then collected andseparated from the eluate using the magnetic separator. The supernatanteluate was transferred to another microfuge tube. This elution step wasrepeated.

[0090] The length and purity of the genomic DNA obtained by thisprocedure was determined by running a sample of the eluted DNA on astandard 1% TAE agarose gel containing ethidium bromide. The yield andpurity were assessed by the ratio of the absorbance at 260 nm (A₂₆₀) tothe absorbance at 280 nm (A₂₈₀) in a spectrophotometer. Typically, thisprocedure yielded 4-8 μg of purified genomic DNA per microfuge tube (200μl whole blood) with an average ratio of A₂₆₀/A₂₈₀ between 1.6 and 1.85.

[0091] Other variations and embodiments of the invention describedherein will now be apparent to those of ordinary skill in the artwithout departing from the spirit of the invention or the scope of theclaims below. All patents, applications, and publications cited in theabove text are incorporated herein by reference.

1. A porous, ferro- or ferrimagnetic, glass particle comprising silicaglass precipitated or adsorbed on iron oxide particles or pigments,wherein the pores of the silica glass particles comprise pores having adiameter greater than and less than 10 nm, and wherein the cumulativepore area of the pores having a diameter greater than 10 nm is greaterthan 4 m²/g.
 2. The porous, ferro- or ferrimagnetic, glass particleaccording to claim 1 having a diameter of about 5-25 μm.
 3. The porous,ferro- or ferrimagnetic, glass particle according to claim 1 having adiameter of about 6-15 μm.
 4. The porous, ferro- or ferrimagnetic, glassparticle according to claim 1 having a diameter of about 7-10 μm.
 5. Theporous, ferro- or ferrimagnetic, glass particle according to claim 1,wherein the total BET surface area of the particles is greater than 190m²/g.
 6. The porous, ferro- or ferrimagnetic, glass particle accordingto claim 1, wherein the total BET surface area of the particle is in therange of about 190-270 m²/g.
 7. The porous, ferro- or ferrimagnetic,glass particle according to claim 1, wherein the cumulative pore area ofthe pores having a diameter greater than 10 nm is in the range of about4-8 m²/g.
 8. The porous, ferro- or ferrimagnetic, glass particleaccording to claim 1, wherein the iron oxide particle or pigment isferrite.
 9. The porous, ferro- or ferrimagnetic, glass particleaccording to claim 1, wherein the iron oxide particle or pigment ismagnetite.
 10. The porous, ferro- or ferrimagnetic, glass particleaccording to claim 1, wherein the iron oxide particle or pigment is amixture of ferrite and magnetite.
 11. The porous, ferro- orferrimagnetic, glass particle according to claim 1, wherein the ironoxide particle or pigment is ferrimagnetic magnetite.
 12. The porrous,ferro- or ferrimagnetic, glass particle according to claim 1, whereinthe particles are about 30-50% by weight SiO₂ and about 50-70% by weightFe₃O₄.
 13. The porrous, ferro- or ferrimagnetic glass particle accordingto claim 12, wherein the particles are about 35-40% by weight SiO₂ andabout 55-65% by weight Fe₃O₄.
 14. The porrous, ferro- or ferrimagneticglass particle according to claim 1, wherein the iron oxide particles orpigments have an average diameter in the range of about 75-300 nm. 15.The porrous, ferro- or ferrimagnetic glass particle according to claim1, wherein 80% of the iron oxide particles or pigments have a diameterin the range of about 75-300 nm.
 16. The porrous, ferro- orferrimagnetic, glass particle according to claim 1, wherein 90% of theiron oxide particles or pigments have a diameter in the range of about75-300 nm.
 17. A kit for separating, isolating, or detecting a moleculeof interest from a mixture comprising a porous, ferro- or ferrimagnetic,glass particle according to any one of claims 1-16.
 18. The kitaccording to claim 17, wherein the molecule of interest is selected fromthe group consisting of nucleic acids, proteins, polypeptides, peptides,carbohydrates, lipids, and combinations thereof.
 19. The kit accordingto claim 18, wherein the molecule of interest is a nucleic acidmolecule.
 20. The kit according to claims 17, 18, or 19 furthercomprising a chaotropic agent.
 21. The kit according to claim 20,wherein the chaotropic agent is selected from the group consisting ofsodium perchlorate, guanidinium hydrochloride, guanidiniumisothiocyanate, potassium iodide, potassium thiocyanate, sodiumchloride, sodium isothiocyanate magnesium chloride, sodium iodide, andcombinations thereof.
 22. A method of making porous, ferro- orferrimagnetic, glass particles comprising: a) providing a suspension offerro- or ferrimagnetic iron oxide particles, b) adding atetraalkoxysilane to the suspension of the ferro- or ferrimagnetic ironoxide particles at a pH between 6 and 8, c) hydrolyzing thetetraalkoxysilane and precipitating silica glass on the ferro- orferrimagnetic iron oxide particles by adding a buffer having a pH lowerthan 6 or higher than 8 to the suspension to form ferro- orferrimagnetic, porous glass particles, d) separating the resultingporous, ferro- or ferrimagnetic, glass particles from suspension e)washing the separated porous, ferro- or ferrimagnetic, glass particleswith an anhydrous alcohol, and f) drying the porous, ferro- orferrimagnetic, glass particles at a temperature below the Curietemperature.
 23. The method of making porous, ferro- or ferrimagnetic,glass particles according to claim 22, wherein the ferro- orferrimagnetic iron oxide particles are provided in a suspension ofglycerol or glycol.
 24. The method of making the porous, ferro- orferrimagnetic, glass particles according to claim 22, wherein the ironoxide particles in suspension range in size from about 75 mn to 300 nm.25. The method of making porous, ferro- or ferrimagnetic, glassparticles according to claim 22, wherein the tetraalkoxysilanecorresponds to the general formula Si(OC_(n)H_(2n+1))₄, where n is 1, 2,3, 4, or
 5. 26. The method of making porous, ferro- or ferrimagnetic,glass particles according to claim 22, wherein the tetraalkoxysilane istetraethoxysilane
 27. The method of making ferro- or ferrimagnetic,porous, glass particles according to claim 22, wherein thetetraalkoxysilane is added to the ferro- or ferrimagnetic iron oxideparticles as a solution of tetraethoxysilane (30%) and an aliphaticC₁-C₄-alcohol (70%).
 28. The method of making porous, ferro- orferrimagnetic, glass particles according to claim 22, wherein thetetraalkoxysilane is added to the suspension of the ferro- orferrimagnetic iron oxide particles at a pH of about
 7. 29. The method ofmaking porous, ferro- or ferrimagnetic, glass particles according toclaim 22, wherein the tetraalkoxysilane is added to the suspension ofthe ferro- or ferrimagnetic iron oxide particles within about tenminutes.
 30. The method of making porous, ferro- or ferrimagnetic, glassparticles according to claim 22, wherein the buffer having a pH lowerthan 6 is an acetate buffer having a pH of about
 5. 31. The method ofmaking porous, ferro- or ferrimagnetic, glass particles according toclaim 22, wherein the buffer having a pH greater than 9 is anammonia/ammonium salt buffer having a pH of about
 11. 32. The method ofmaking porous, ferro- or ferrimagnetic, glass particles according toclaim 22, wherein the anhydrous alcohol used to wash the particles isanhydrous ethanol.
 33. The method of making the porous, ferro- orferrimagnetic, glass particles according to claim 22, wherein thetemperature at which the particles are dried is about 300° C.
 34. Amethod of making porous, ferro- or ferrimagnetic, glass particlescomprising: a) providing a suspension of ferro- or ferrimagnetic ironoxide particles in a glycerol solution that has a pH of 5 or less or apH of 9 or greater, b) adding a tetraethyoxysilane dropwise withstirring to the suspension of the ferro- or ferrimagnetic iron oxideparticles within 10 minutes, c) aging the mixture to allow the ferro- orferrimagnetic porous glass particles to solidify and stabilize, d)separating the resulting porous, ferro- or ferrimagnetic, glassparticles from the liquid by applying an external magnetic field, e)washing the separated porous, ferro- or ferrimagnetic, glass particleswith an anhydrous alcohol, and f) dying the porous, ferro- orferrimagnetic, glass particles at a temperature between about 300 and500° C.
 35. A method of Dung porous, ferro- or ferrimagnetic, glassparticles comprising: a) providing a suspension of ferro- orferrimagnetic iron oxide particles in alcohol, preferably ethanol, b)adding silica nanoparticles to the suspension of the ferro- orferrimagnetic iron oxide particles at a pH lower than 6, c) aging themixture by continuous stirring, d) separating the resulting porous,ferro- or ferrimagnetic, glass particles from the liquid, e) washing theseparated porous, ferro- or ferrimagnetic, glass particles, and f)drying the porous, ferro- or ferrimagnetic, glass particles at atemperature at about 200° C.
 36. A method of making porous, ferro- orferrimagnetic, glass particles comprising: a) providing a suspension offerro- or ferrimagnetic iron oxide particles in paraffin oil b) addingan aquaeous sodium silicate solution, preferably 27% SiO₂ in water, tothe suspension of the ferro- or ferrimagnetic iron oxide particles at apH lower than 6, c) aging the mixture by continuous stirring, d)separating the resulting porous, ferro- or ferrimagnetic, glassparticles from the liquid, e) washing the separated porous, ferro- orferrimagnetic, glass particles, and f) drying the porous, ferro- orferrimagnetic, glass particles at a temperature at about 200° C.
 37. Amethod of isolating a molecule of interest from a mixture, comprising:providing a mixture containing a molecule of interest; contacting themixture with a porous, ferro- or ferrimagnetic, glass particle of anyone of claims 1-16; allowing the molecule of interest in the mixture toadhere to the porous, ferro- or ferrimagnetic, glass particles;collecting the porous, ferro- or ferrimagnetic, glass particlescontaining the adherent molecule of interest by applying an externalmagnetic field; and removing the porous, ferro- or ferrimagnetic, glassparticles with the adherent molecule of interest from the unboundcomponents of the mixture.
 38. The method of isolating a molecule ofinterest from a mixture according to claim 37, further comprising thestep of eluting the adherent molecule of interest from the porous,ferro- or ferrimagnetic, glass particles.
 39. The method of isolating amolecule of interest from a mixture according to claim 37 or claim 38,wherein the molecule of interest is selected from the group consistingof nucleic acids, proteins, polypeptides, peptides, carbohydrates,lipids, and combinations thereof.
 40. The method of isolating a moleculeof interest from a mixture according to claim 39 wherein the molecule ofinterest is a nucleic acid molecule.
 41. The method of isolating anucleic acid according to claim 40, wherein the nucleic acid is selectedfrom the group consisting of plasmid DNA, genomic DNA, cDNA, PCR DNA,linear DNA, RNA, ribozymes, aptamers, and chemically synthesized nucleicacids.